The 31P-NMR technique of magnetization transfer can be used to investigate the intracellular kinetics of the creatine kinase reaction explicitly in isolated rat heart and in skeletal muscle of the hindlimb of the living rat. The purpose of this research project is to characterize the creatine kinase reaction in vivo. Validation studies will focus on the regulation of creatine kinase activity as pH, temperature and creatine phosphate content are altered. The former two experiments will be performed in isolated rat hearts perfused with media at various pH values or at various temperatures. The latter experiment will be carried out in isolated hearts and in the skeletal muscle of rats that have been fed a diet that contains 1% Beta-guanidinoproprionic acid for 6 weeks. Preliminary studies from our laboratory suggest that creatine kinase flux in the isolated, well-oxygenated rat hearts increases with cardiac performance, oxygen consumption and mitochondrial respiration, thus providing in vivo evidence for the creatine phosphate shuttle. Further investigation will evaluate the effect of the inotropic agent norepinephrine on creatine kinase flux, cardiac performance and mitochondrial respiration in the isolated rat heart. Also, the relation between muscle contraction and creatine kinase flux will be investigated in the quadriceps muscle of the living rat by correlating creatine kinase flux, measured with a 31P-NMR surface coil, to the rate of muscle contraction from stimulation of the femoral nerve. 31P-NMR will be used to measure creatine kinase flux in the hypoxic rat heart. Correlating flux to cardiac performance and to oxygen consumption in hypoxic hearts will determine whether creatine kinase flux is coupled to the rate of high-energy phosphate utilization, as reflected in cardiac performance, or to the rate of mitochondrial ATP production, as estimated by oxygen consumption. Other studies will identify the relationship among creatine kinase flux, cardiac performance and oxygen consumption in hearts that have been reperfused after ischemia in order to determine whether cardiac performance in post-ischemic rat hearts is depressed because of a disruption or uncoupling between mitochondrial respiration and creatine kinase flux. In addition to providing critical information about enzyme activity in vivo and muscle bioenergetics, the experiments proposed here will contribute to the understanding of high-energy phosphate metabolism needed for the clinical application of 31P-NMR.